"The Fas/FasL apoptotic pathway is important in tumor cell growth and progression. We have studied this pathway in neuroblastoma (NB) and the Ewing sarcoma family of tumors (ESFT) in order to understand the molecular events that lead to impaired programmed cell death (apoptosis) in these tumors. The objective of this project is to identify the reasons for treatment failure in NB and ESFT and possibly find ways to manipulate the Fas/FasL system for therapeutic purposes. Our material consists of established human tumor cell lines and paraffin-embedded tumor tissue sections. The methods employed for these studies consist of in vitro cytotoxic assays, DNA fragmentation ELISA, TUNEL, electron microscopy, immunostaining, Western blotting, reverse transcription polymerase chain reaction (RT-PCR), Northern blotting and DNA sequencing. Our data have shown that NB and ESFT express Fas and FasL. However, the expression and ligation of the Fas receptor is not sufficient to drive tumor cells to suicide, because not all tumor cells possess a functional Fas signaling pathway. Specifically, we found that the apoptotic signal is transmitted in ESFT, but not in NB cell lines, despite an initial engagement of the Fas receptor in the latter. Our finding that an exogenously administered synthetic ceramide analog induced bcl-2 downregulation, caspase 3 activation and apoptosis in NB, suggested that the apoptotic signal is blocked at the mitochondrial level in this tumor and that bcl-2 may play an important inhibitory role. In the Fas-responsive ESFT cell lines, we found that we can induce apoptosis not only by ligation with a Fas activating antibody, but also by upregulation of the expression of Fas and FasL on the cell surface in the absence of increased synthesis. Increased surface expression of Fas and FasL was accomplished after treatment with synthetic metalloproteinase inhibitors (MMPIs) which inhibit the cleavage of the full length FasL molecule to its soluble form. In addition to inducing apoptotis, MMPIs sensitized ESFT cells to the chemotherapeutic agent doxorubicin.These findings support that MMPIs may play a role in the treatment of Fas-sensitive ESFT, possibly in combination treatments with traditional chemotherapeutic agents."